Cell-derived viral vaccines with low levels of residual cell DNA

ABSTRACT

The present invention relates to vaccine products for the treatment or prevention of viral infections. Further provided are methods of reducing contaminants associated with the preparation of cell culture vaccines. Residual functional cell culture DNA is degraded by treatment with a DNA alkylating agent, such as β-propiolactone (BPL), thereby providing a vaccine comprising immunogenic proteins derived from a virus propagated on cell culture, substantially free of residual functional cell culture DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/092,190, filed Sep. 3, 2008, which is the U.S. National Phase entryunder 35 U.S.C. § 371 of International Patent Application No.PCT/IB2006/003880, filed Nov. 1, 2006, which claims benefit of priorityto U.S. Provisional Patent Application No. 60/732,786, filed Nov. 1,2005, all of which are incorporated herein by reference in theirentirety.

All documents and on-line information cited herein are incorporated byreference in their entirety.

TECHNICAL FIELD

The invention provides improved cell culture products and processes withreduced impurities. Specifically, the invention provides an improvedmethod of degrading any residual functional cell culture DNA remainingassociated with the cell culture generated product. According to theinvention, residual functional cell culture DNA is degraded by treatmentwith a DNA alkylating agent, such as β-propiolactone (BPL). This processmay be used to treat a range of cell culture products including vaccinesand recombinant proteins.

BACKGROUND ART

Commercial production of viral vaccines typically require largequantities of virus as an antigen source. Commercial quantities of virusfor vaccine production may be achieved by culture and replication of aseed virus in a cell culture system. Cell culture systems suitable forviral replication include mammalian, avian or insect cells, butmammalian cell culture systems are particularly preferred for viralvaccines to ensure proper glycosylation and folding of the virus'antigens proteins. For similar reasons, mammalian cell culture systemsare also preferred for recombinant protein expression.

If unmodified from their naturally occurring states, cell cultures havea limited ability to reproduce, and subsequently are impractical andinefficient for producing the amount of material necessary for acommercial vaccine or recombinant protein. Consequently, formanufacturing purposes, it is preferred that the cells are modified tobe “continuous” or “immortalized” cell lines to increase the number oftimes they can divide. Many of these modifications employ mechanismssimilar to those which are implicated in oncogenic cells. As such, thereis a concern that any residual materials from the cell culture process,such as host cell DNA, be removed from the final formulation of avaccine or recombinant protein product manufactured in these systems.

A standard way of removing residual host cell DNA is by DNase treatment.A convenient method of this type is disclosed in European patent 0870508and U.S. Pat. No. 5,948,410, involving a two-step treatment, first usinga DNase (e.g. Benzonase) and then a cationic detergent (e.g. CTAB).

Current efforts to reduce this risk have focused on reducing the totalconcentration of residual host cell DNA. It is an object of the presentinvention to reduce the risk further by eliminating the functionality ofany remaining host cell DNA.

SUMMARY OF THE INVENTION

The invention provides improved cell culture products and processes withreduced impurities. Specifically, the invention provides an improvedmethod of degrading any residual functional cell culture DNA remainingassociated with the cell culture generated product. According to theinvention, residual functional cell culture DNA is degraded by treatmentwith a DNA alkylating agent, such as β-propiolactone (BPL). This processmay be used to treat a range of cell culture products including vaccinesand recombinant proteins.

The invention includes a vaccine comprising immunogenic proteins derivedfrom a virus propagated on cell culture, wherein the vaccine issubstantially free of residual functional cell culture DNA. In addition,the invention relates to recombinant proteins expressed in cell culture,where the final recombinant protein formulation is substantially free ofresidual functional cell culture DNA.

Functionality of any residual host cell DNA may be eliminated bytreatment of the DNA with an alkylating agent which cleaves the DNA intoportions small enough so that it is unable to code for a functionalprotein, to be transposed into a human recipient's chromosome, orotherwise recognized by recipient DNA replication machinery. Preferably,the length of degraded residual cell culture DNA is less than 500 basepairs. More preferably, the length of degraded residual cell culture DNAis less than 200 base pairs.

DETAILED DESCRIPTION

The invention provides improved cell culture products and processes withreduced impurities. Specifically, the invention provides an improvedmethod of degrading any residual functional cell culture DNA remainingassociated with the cell culture generated product. According to theinvention, residual functional cell culture DNA is degraded by treatmentwith a DNA alkylating agent, such as β-propiolactone (BPL). This processmay be used to treat a range of cell culture products including vaccinesand recombinant proteins.

The invention includes a vaccine comprising immunogenic proteins derivedfrom a virus propagated on cell culture, wherein the vaccine issubstantially free of residual functional cell culture DNA. In addition,the invention relates to recombinant proteins expressed in cell culture,where the final recombinant protein formulation is substantially free ofresidual functional cell culture DNA.

Functionality of any residual host cell DNA may be eliminated bytreatment of the DNA with an alkylating agent which cleaves the DNA intoportions small enough so that it is unable to code for a functionalprotein, to be transposed into a human recipient's chromosome, orotherwise be recognized by recipient DNA replication machinery. Thelength of degraded (non-functional) residual cell culture DNA ispreferably less than 1000 base pairs (e.g. less than 1000, 800, 700,600, 500, 400, 300, 200, 150, 100, 75, or 50 base pairs). Preferably,the length of degraded residual cell culture DNA is less than 500 basepairs. More preferably, the length of degraded residual cell culture DNAis less than 200 base pairs.

As used herein, reference to “functional DNA” or “functional RNA”indicates a nucleotide sequence capable of being translated into afunctional protein or transposed into a mammalian chromosome. Generally,nucleotide sequences capable of being translated to a functional proteinrequire promoter regions, start codons, stop codons, and internal codingsequences for functional proteins. Where DNA damage occurs, as fromaddition of an alkylating agent, many of these regions are altered ordestroyed, such that translation can longer proceed or only proceeds toform an oligopeptide subunit of the intended protein.

“Degraded residual functional cell culture DNA” refers to functional DNAthat cannot be translated into a functional protein or transposed into amammalian chromosome. Preferably, “degraded residual functional DNA” hasa length of less than 1000 base pairs, more preferably less than 500base pairs, even more preferably less than 250 base pairs, and mostpreferably less than 100 base pairs. The length of the degraded residualfunctional DNA can be determined by standard techniques, including gelelectrophoresis.

The invention provides for vaccine compositions and recombinant proteinformulations which are substantially free of residual functional cellculture DNA. As used herein, substantially free of residual functionalcell culture DNA refers to a composition or formulation where residualDNA fragments of less than 200 basepairs are detectable at less than 10ng per 0.5 ml. The size of any residual cell culture DNA may be measuredby standard techniques, including capillary gel electrophoresis andnucleic acid amplification technology.

The use of an alkylating agent such as BPL in the invention provides theadditional benefit of reducing aggregation and contaminants. Vaccineformulations with reduced aggregates may also have improvedimmunogenicity. Immunogenicity of a vaccine relies upon specificity ofantibodies for particular viral epitopes. If the surface of a protein isbound or masked by unwanted molecules or hidden through aggregation inlarge macromolecules, the epitopes may become less recognizable andtherefore less efficacious in a vaccine. In addition, vaccineformulations with reduced aggregates may have additional processingadvantages. Purification processes rely upon isolation of the expectedprotein, for example hemagglutinin and neuraminidase in an influenzavaccine. If the protein is structurally modified by the presence ofaggregates or cross-linking it may not be recognized and subsequentlyremoved by column chromatography, filtration, or centrifugation.

Alkylating Agents

Alkylating agents for use in the invention include substances thatintroduce an alkyl radical into a compound. Preferably, the alkylatingagent is a monoalkylating agent, such as BPL. BPL is a monoalkylatingagent widely used for inactivation of viruses in the preparation of manyvaccines. BPL reacts with various biological molecules including nucleicacids where it causes structural modification by alkylation anddepurination. BPL is generally represented by the following structure:

In vitro, BPL generally reacts with nucleophiles present in highconcentrations, under conditions favorable to nucleophilic substitutionreactions—as in high heat, high BPL concentrations, and aprotic polarsolvents—to form functionalized propionic acids, such as7-(2-carboxyethyl)guanine or 1-(2-carboxyethyl)deoxyadenosine (Scheme1). Boutwell et al. Annals New York Academy of Sciences, 751-764; Perrinet al. Biologicals, 23 (1995) 207-211; Chen et al. Carcinogenesis, 2(2)(1981) 73-80.

Such binding or alkylating of DNA bases induces mutagenicity by a numberof mechanisms including base pair substitutions, especiallydepurination, deletions, and cross linking of nucleosides. The highdegree of mutagenicity and reactivity of BPL corresponds to rapid viralkilling and subsequent DNA degradation to non-carcinogenic by-products.

Any residual functional cell culture DNA is degraded by treatment withless than 1% BPL (e.g. less than 1%, 0.75%, 0.5%, 0.25%, 0.2%, 0.1%,0.075%, 0.05%, 0.025%, 0.01%, or 0.005%). Preferably, residualfunctional cell culture DNA is degraded by treatment with between 0.1%and 0.01% BPL.

The alkylating agent is preferably added to a buffered solution and thepH of the solution is preferably maintained between 5 and 10. Morepreferably the pH of the solution is maintained between 6 and 9. Evenmore preferably the pH of the solution is maintained between 7 and 8.

In some methods, the alkylating agent is added more than once. Forinstance, a first BPL treatment may be performed and then a second BPLtreatment may be performed. Between the first and second treatmentsthere may be an alkylating agent removal step, but the alkylating agentmay be added for the second treatment without removing any alkylatingagent remaining from the first treatment.

Preferably, the alkylating agent is also used as the inactivating agentfor the virus used in the vaccine. The alkylating agents of theinvention are preferred over traditional inactivating agents, such asformaldehyde, which can cross-link proteins to other material, includinghost cell DNA. Such cross-linking can lead to the formation ofaggregates (such as protein-protein conglomerates, nucleotidecombinations, and protein-nucleotide combinations). Because alkylatingagents, such as BPL, do not rely on such cross-linking mechanisms forviral inactivation, use of such alkylating agents for viral inactivationminimizes the formation of aggregates and other impurities in thevaccine product. Such aggregates may comprises proteins ionically orcovalently bound to other proteins, proteins ionically or covalentlybound to other nucleotides, and/or nucleotides ionically or covalentlybound to other nucleotides.

As used herein, reference to “aggregation” or “an aggregate” indicates amass or body of individual units or particles bound together to createlarger groups or particles. Aggregation can be generally be determinedby quantitative measurements of the desired components before and aftera possible aggregating step or before and after application of anaggregate-disrupting method (e.g. by detergent treatment), by gelelectrophoresis (such as Laemmli's system), chromatography, solutionturbidity, or sedimentation studies and other methods well known in theart.

Treatment with the alkylating agent, particularly with BPL, may involvephases with different temperatures. For instance, there may be a firstphase at a low temperature (e.g. at between 2-8° C., such as about 4°C.) and a second phase at a higher temperature, typically at least 10°C. higher than the first phase (e.g. at between 25-50° C., such as about37° C.). This two-phase process is particularly useful where thealkylating agent is being used for both inactivation and DNAdegradation. In a typical scheme, virus inactivation occurs during thelower temperature phase, and DNA degradation occurs during the highertemperature phase. As described in more detail below, an increasedtemperature can also facilitate removal of a heat-sensitive alkylatingreagent.

Immunogenic Proteins

Immunogenic proteins suitable for use in the invention may be derivedfrom any virus which is the target of a vaccine. The immunogenicproteins may be formulated as inactivated (or killed) virus, attenuatedvirus, split virus formulations, purified subunit formulations, viralproteins which are isolated, purified or derived from a virus, and viruslike particles (VLPs).

The immunogenic proteins of the invention are viral antigens whichpreferably include epitopes which are exposed on the surface of thevirus during at least one stage of its life cycle. The viral antigensare preferably conserved across multiple serotypes or isolates. Theviral antigens include antigens derived from one or more of the virusesset forth below as well as the specific antigens examples identifiedbelow. Viruses may be non-enveloped or, preferably, enveloped. Virusesare preferably RNA viruses, and more preferably ssRNA viruses. They mayhave a sense or, preferably, an antisense genome. Their genomes may benon-segmented or, preferably, segmented.

Orthomyxovirus: Viral antigens may be derived from an Orthomyxovirus,such as Influenza A, B and C. Orthomyxovirus antigens may be selectedfrom one or more of the viral proteins, including hemagglutinin (HA),neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membraneprotein (M2), one or more of the transcriptase components (PB1, PB2 andPA). Preferred antigens include HA and NA.

Influenza antigens may be derived from interpandemic (annual) flustrains. Alternatively influenza antigens may be derived from strainswith the potential to cause pandemic a pandemic outbreak (i.e.,influenza strains with new hemagglutinin compared to a hemagglutinin incurrently circulating strains, or influenza strains which are pathogenicin avian subjects and have the potential to be transmitted horizontallyin the human population, or influenza strains which are pathogenic tohumans). Depending on the particular season and on the nature of theantigen included in the vaccine, the influenza antigens may be derivedfrom one or more of the following hemagglutinin subtypes: H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.

The influenza antigens of the invention may be derived from an avianinfluenza strain, particularly a highly pathogenic avian influenzastrain (HPAI). Alexander, Avian Dis (2003) 47(3 Suppl):976-81).

Further details of influenza virus antigens are given below.

Paramyxoviridae viruses: Viral antigens may be derived fromParamyxoviridae viruses, such as Pneumoviruses (RSV), Paramyxoviruses(PIV) and Morbilliviruses (Measles).

Pneumovirus: Viral antigens may be derived from a Pneumovirus, such asRespiratory syncytial virus (RSV), Bovine respiratory syncytial virus,Pneumonia virus of mice, and Turkey Rhinotracheitis virus. Preferably,the Pneumovirus is RSV. Pneumovirus antigens may be selected from one ormore of the following proteins, including surface proteins Fusion (F),Glycoprotein (G) and Small Hydrophobic protein (SH), matrix proteins Mand M2, nucleocapsid proteins N, P and L and nonstructural proteins NS1and NS2. Preferred Pneumovirus antigens include F, G and M. See e.g., JGen Virol. 2004 November; 85(Pt 11):3229). Pneumovirus antigens may alsobe formulated in or derived from chimeric viruses. For example, chimericRSV/PIV viruses may comprise components of both RSV and PIV.

Paramyxovirus: Viral antigens may be derived from a Paramyxovirus, suchas Parainfluenza virus types 1-4 (PIV), Mumps, Sendai viruses, Simianvirus 5, Bovine parainfluenza virus and Newcastle disease virus.Preferably, the Paramyxovirus is PIV or Mumps. Paramyxovirus antigensmay be selected from one or more of the following proteins:Hemagglutinin-Neuraminidase (HN), Fusion proteins F1 and F2,Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrixprotein (M). Preferred Paramyxovirus proteins include HN, F1 and F2.Paramyxovirus antigens may also be formulated in or derived fromchimeric viruses. For example, chimeric RSV/PIV viruses may comprisecomponents of both RSV and PIV. Commercially available mumps vaccinesinclude live attenuated mumps virus, in either a monovalent form or incombination with measles and rubella vaccines (MMR).

Morbillivirus: Viral antigens may be derived from a Morbillivirus, suchas Measles. Morbillivirus antigens may be selected from one or more ofthe following proteins: hemagglutinin (H), Glycoprotein (G), Fusionfactor (F), Large protein (L), Nucleoprotein (NP), Polymerasephosphoprotein (P), and Matrix (M). Commercially available measlesvaccines include live attenuated measles virus, typically in combinationwith mumps and rubella (MMR).

Picornavirus: Viral antigens may be derived from Picornaviruses, such asEnteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses andAphthoviruses. Antigens derived from Enteroviruses, such as Poliovirusare preferred.

Enterovirus: Viral antigens may be derived from an Enterovirus, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71. Preferably, the Enterovirusis Poliovirus. Enterovirus antigens are preferably selected from one ormore of the following Capsid proteins VP1, VP2, VP3 and VP4.Commercially available polio vaccines include Inactivated Polio Vaccine(IPV) and Oral poliovirus vaccine (OPV).

Heparnavirus: Viral antigens may be derived from an Heparnavirus, suchas Hepatitis A virus (HAV). Commercially available HAV vaccines includeinactivated HAV vaccine.

Togavirus: Viral antigens may be derived from a Togavirus, such as aRubivirus, an Alphavirus, or an Arterivirus. Antigens derived fromRubivirus, such as Rubella virus, are preferred. Togavirus antigens maybe selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. Togavirusantigens are preferably selected from E1, E2 or E3. Commerciallyavailable Rubella vaccines include a live cold-adapted virus, typicallyin combination with mumps and measles vaccines (MMR).

Flavivirus: Viral antigens may be derived from a Flavivirus, such asTick-borne encephalitis (TBE), Dengue (types 1, 2, 3 or 4), YellowFever, Japanese encephalitis, West Nile encephalitis, St. Louisencephalitis, Russian spring-summer encephalitis, Powassan encephalitis.Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a,NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens are preferablyselected from PrM, M and E. Commercially available TBE vaccine includeinactivated virus vaccines.

Pestivirus: Viral antigens may be derived from a Pestivirus, such asBovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Borderdisease (BDV).

Hepadnavirus: Viral antigens may be derived from a Hepadnavirus, such asHepatitis B virus. Hepadnavirus antigens may be selected from surfaceantigens (L, M and S), core antigens (HBc, HBe). Commercially availableHBV vaccines include subunit vaccines comprising the surface antigen Sprotein.

Hepatitis C virus: Viral antigens may be derived from a Hepatitis Cvirus (HCV). HCV antigens may be selected from one or more of E1, E2,E1/E2, NS345 polyprotein, NS 345-core polyprotein, core, and/or peptidesfrom the nonstructural regions (Houghton et al., Hepatology (1991)14:381).

Rhabdovirus: Viral antigens may be derived from a Rhabdovirus, such as aLyssavirus (Rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigensmay be selected from glycoprotein (G), nucleoprotein (N), large protein(L), nonstructural proteins (NS). Commercially available Rabies virusvaccine comprise killed virus grown on human diploid cells or fetalrhesus lung cells.

Caliciviridae; Viral antigens may be derived from Calciviridae, such asNorwalk virus, and Norwalk-like Viruses, such as Hawaii Virus and SnowMountain Virus.

Coronavirus: Viral antigens may be derived from a Coronavirus, SARS,Human respiratory coronavirus, Avian infectious bronchitis (TRV), Mousehepatitis virus (MHV), and Porcine transmissible gastroenteritis virus(TGEV). Coronavirus antigens may be selected from spike (S), envelope(E), matrix (M), nucleocapsid (N), and Hemagglutinin-esteraseglycoprotein (HE). Preferably, the Coronavirus antigen is derived from aSARS virus. SARS viral antigens are described in WO 04/92360.

Retrovirus: Viral antigens may be derived from a Retrovirus, such as anOncovirus, a Lentivirus or a Spumavirus. Oncovirus antigens may bederived from HTLV-1, HTLV-2 or HTLV-5. Lentivirus antigens may bederived from HIV-1 or HIV-2. Retrovirus antigens may be selected fromgag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigensmay be selected from gag (p24gag and p55gag), env (gp160 and gp41), pol,tat, nef, rev vpu, miniproteins, (preferably p55 gag and gp140v delete).HIV antigens may be derived from one or more of the following strains:HIV_(IIIb), HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235),HIV-1_(US4).

Reovirus: Viral antigens may be derived from a Reovirus, such as anOrthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Reovirusantigens may be selected from structural proteins λ1, λ2, λ3, μ1, μ2,σ1, σ2, or σ3, or nonstructural proteins σNS, μNS, or σ1s. PreferredReovirus antigens may be derived from a Rotavirus. Rotavirus antigensmay be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 andVP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Preferred Rotavirusantigens include VP4 (or the cleaved product VP5 and VP8), and VP7.

Parvovirus: Viral antigens may be derived from a Parvovirus, such asParvovirus B19. Parvovirus antigens may be selected from VP-1, VP-2,VP-3, NS-1 and NS-2. Preferably, the Parvovirus antigen is capsidprotein VP-2.

Delta hepatitis virus (HDV): Viral antigens may be derived HDV,particularly δ-antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814).

Hepatitis E virus (HEV): Viral antigens may be derived from HEV.

Hepatitis G virus (HGV): Viral antigens may be derived from HGV.

Human Herpesvirus: Viral antigens may be derived from a HumanHerpesvirus, such as Herpes Simplex Viruses (HSV), Varicella-zostervirus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), HumanHerpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus8 (HHV8). Human Herpesvirus antigens may be selected from immediateearly proteins (α), early proteins (β) and late proteins (γ). HSVantigens may be derived from HSV-1 or HSV-2 strains. HSV antigens may beselected from glycoproteins gB, gC, gD and gH, fusion protein (gB), orimmune escape proteins (gC, gE, or gI). VZV antigens may be selectedfrom core, nucleocapsid, tegument, or envelope proteins. A liveattenuated VZV vaccine is commercially available. EBV antigens may beselected from early antigen (EA) proteins, viral capsid antigen (VCA),and glycoproteins of the membrane antigen (MA). CMV antigens may beselected from capsid proteins, envelope glycoproteins (such as gB andgH), and tegument proteins.

Papovaviruses: Antigens may be derived from Papovaviruses, such asPapillomaviruses and Polyomaviruses. Papillomaviruses include HPVserotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47,51, 57, 58, 63 and 65. Preferably, HPV antigens are derived fromserotypes 6, 11, 16 or 18. HPV antigens may be selected from capsidproteins (L1) and (L2), or E1-E7, or fusions thereof. HPV antigens arepreferably formulated into virus-like particles (VLPs). Polyomyavirusviruses include BK virus and JK virus. Polyomavirus antigens may beselected from VP1, VP2 or VP3.

Further provided are viral antigens described in Vaccines, 4^(th)Edition (Plotkin and Orenstein ed. 2004); Medical Microbiology 4^(th)Edition (Murray et al. ed. 2002); Virology, 3rd Edition (W. K. Jokliked. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M.Knipe, eds. 1991), which are contemplated in conjunction with thecompositions of the present invention.

Immunogenic proteins suitable for use in the invention may be derivedfrom a virus which causes respiratory disease. Examples of suchrespiratory antigens include proteins derived from a respiratory virussuch as Orthomyxoviruses (influenza), Pneumovirus (RSV), Paramyxovirus(PIV), Morbillivirus (measles), Togavirus (Rubella), VZV, andCoronavirus (SARS). Immunogenic proteins derived from influenza virusare particularly preferred.

The compositions of the invention may include one or more immunogenicproteins suitable for use in pediatric subjects. Pediatric subjects aretypically less than about 3 years old, or less than about 2 years old,or less than about 1 years old. Pediatric antigens may be administeredmultiple times over the course of 6 months, 1, 2 or 3 years. Pediatricantigens may be derived from a virus which may target pediatricpopulations and/or a virus from which pediatric populations aresusceptible to infection. Pediatric viral antigens include antigensderived from one or more of Orthomyxovirus (influenza), Pneumovirus(RSV), Paramyxovirus (PIV and Mumps), Morbillivirus (measles), Togavirus(Rubella), Enterovirus (polio), HBV, Coronavirus (SARS), andVaricella-zoster virus (VZV), Epstein Barr virus (EBV).

The compositions of the invention may include one or more immunogenicproteins suitable for use in elderly or immunocompromized individuals.Such individuals may need to be vaccinated more frequently, with higherdoses or with adjuvanted formulations to improve their immune responseto the targeted antigens. Antigens which may be targeted for use inElderly or Immunocompromized individuals include antigens derived fromone or more of the following viruses: Orthomyxovirus (influenza),Pneumovirus (RSV), Paramyxovirus (PIV and Mumps), Morbillivirus(measles), Togavirus (Rubella), Enterovirus (polio), HBV, Coronavirus(SARS), Varicella-zoster virus (VZV), Epstein Barr virus (EBV), andCytomegalovirus (CMV).

After growing viruses, the aklylating agent may be used on purifiedvirions e.g. on virions present in a clarified cell culture, or onvirions purified from such a clarified cell culture. A method of theinvention may involve removing cellular material by clarification, andthen purification of virions from the clarified cell culture e.g. bychromatography. The alkylating agent maybe used on virions purified inthis manner, or after a further optional step ofultrafiltration/diafiltration. Preferred methods use the aklylatingagent not on the clarified supernatant of an infected cell culture, buton virions purified from such a clarified supernatant (cf. Morgeaux etal. (1993) Vaccine 11:82-90).

The aklylating agent is preferably used after a step of endotoxinremoval has taken place.

Method Steps

The vaccine compositions of the invention may be prepared by isolationof the immunogenic protein and degradation of residual functional hostcell DNA. Similarly, recombinant protein formulations may be prepared byisolation or purification of the recombinant protein and degradation ofresidual functional host cell DNA. These steps may be carried outsequentially or simultaneously. The step of degrading residualfunctional cell culture DNA is done by addition of an alkylating agente.g. BPL.

The alkylating agent and any residual side products are preferablyremoved prior to the final formulation of the vaccine or recombinantprotein. Preferably, the vaccine composition or recombinant proteinformulation contains less than 0.1% free propionic acid and BPL combined(e.g. less than 0.1%, 0.05%, 0.025%, 0.01%, 0.005%, 0.001%, or 0.01%.Preferably, the final vaccine composition or recombinant proteinformulation contains less than 0.01% BPL.

BPL can conveniently be removed by heating, to cause hydrolysis into thenon-toxic β-hydroxypropionic acid. The length of time required forhydrolysis depends on the total amount of BPL and the temperature.Higher temperatures given more rapid hydrolysis, but the temperatureshould not be raized so high as to damage the active proteinaceousingredients. As described below, heating to about 37° C. for 2-2.5 hoursis suitable for removing BPL.

After treatment of DNA, the residual DNA products may be retained in thefinal immunogenic or recombinant composition. More preferably, however,they are separated from desired components e.g. separated fromvirions/proteins. Separation in this way may remove DNA degradationproducts in part or in full, and preferably removes any degraded DNAthat is >200 bp. Thus a method of the invention may include a step ofseparating DNA from virions. This separation step may involve e.g. oneor more of ultrafiltration, ultracentrifugation (including gradientultracentrifugation), virus core pelleting and supernatantfractionation, chromatography (such as ion exchange chromatography e.g.anion exchange), adsorption, etc.

Overall, therefore, a method may involve treatment with an alkylatingagent to degrade the length of residual DNA, and later purification toremove residual DNA (including removal of degraded DNA).

Cell Culture

Vaccines of the invention are prepared from viruses which are propagatedon cell culture. In addition, the invention includes formulations ofrecombinant proteins expressed in cell culture. Mammalian cell culturesare preferred for both viral replication and recombinant proteinexpression.

A number of mammalian cell lines are known in the art and include celllines derived from human or non-human primate (e.g. monkey) cells (e.g.PER.C6 cells which are described, for example, in WO01/38362,WO01/41814, WO02/40665, WO2004/056979, and WO2005/080556, incorporatedby reference herein in their entireties, as well as deposited underECACC deposit number 96022940), MRC-5 (ATCC CCL-171), WI-38 (ATCCCCL-75), HEK cells, HeLa cells, fetal rhesus lung cells (ATCC CL-160),human embryonic kidney cells (293 cells, typically transformed bysheared adenovirus type 5 DNA), Vero cells (from monkey kidneys), horse,cow (e.g. MDBK cells), sheep, dog (e.g. MDCK cells from dog kidneys,ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 asdescribed in WO 97/37000 and WO 97/37001), cat, and rodent (e.g. hamstercells, such as BHK21-F, HKCC cells, or Chinese hamster ovary (CHO)cells), and may be obtained from a wide variety of developmental stages,including for example, adult, neonatal, fetal, and embryo.

Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line. Suitable dog cells are e.g.kidney cells, as in the MDCK cell line. Thus suitable cell linesinclude, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5;PER.C6; WI-38; etc. The use of mammalian cells means that vaccines canbe free from materials such as chicken DNA, egg proteins (such asovalbumin and ovomucoid), etc., thereby reducing allergenicity.

In certain embodiments the cells are immortalized (e.g. PER.C6 cells;ECACC 96022940). In preferred embodiments, mammalian cells are utilized,and may be selected from and/or derived from one or more of thefollowing non-limiting cell types: fibroblast cells (e.g. dermal, lung),endothelial cells (e.g. aortic, coronary, pulmonary, vascular, dermalmicrovascular, umbilical), hepatocytes, keratinocytes, immune cells(e.g. T cell, B cell, macrophage, NK, dendritic), mammary cells (e.g.epithelial), smooth muscle cells (e.g. vascular, aortic, coronary,arterial, uterine, bronchial, cervical, retinal pericytes), melanocytes,neural cells (e.g. astrocytes), prostate cells (e.g. epithelial, smoothmuscle), renal or kidney cells (e.g. epithelial, mesangial, proximaltubule), skeletal cells (e.g. chondrocyte, osteoclast, osteoblast),muscle cells (e.g. myoblast, skeletal, smooth, bronchial), liver cells,retinal cells or retinoblasts, lung cells, and stromal cells.

WO97/37000 and WO97/37001 describe production of animal cells and celllines that capable of growth in suspension and in serum free media andare useful in the production and replication of viruses, particularlyinfluenza virus. Further details are given in WO03/023021 andWO03/023025.

Preferred mammalian cell lines for growing influenza viruses include:MDCK cells, derived from Madin Darby canine kidney; Vero cells, derivedfrom African green monkey (Cercopithecus aethiops) kidney; or PER.C6cells, derived from human embryonic retinoblasts. These cell lines arewidely available e.g. from the American Type Cell Culture (ATCC)collection, from the Coriell Cell Repositories, or from the EuropeanCollection of Cell Cultures (ECACC). For example, the ATCC suppliesvarious different Vero cells under catalog numbers CCL-81, CCL-81.2,CRL-1586 and CRL-1587, and it supplies MDCK cells under catalog numberCCL-34. PER.C6 is available from the ECACC under deposit number96022940.

The most preferred cell lines for growing influenza viruses are MDCKcell lines. The original MDCK cell line is available from the ATCC asCCL-34, but derivatives of this cell line may also be used. Forinstance, WO97/37000 discloses a MDCK cell line that was adapted forgrowth in suspension culture (‘MDCK 33016’, deposited as DSM ACC 2219).Similarly, EP-A-1260581 (WO01/64846) discloses a MDCK-derived cell linethat grows in suspension in serum-free culture (‘B-702’, deposited asFERM BP-7449). WO2006/071563 discloses non-tumorigenic MDCK cells,including ‘MDCK-S’ (ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501),‘MDCK-SF102’ (ATCC PTA-6502) and ‘MDCK-SF103’ (PTA-6503). WO2005/113758discloses MDCK cell lines with high susceptibility to infection,including ‘MDCK.5F1’ cells (ATCC CRL-12042). Any of these MDCK celllines can be used.

Manipulation of MDCK cell cultures in suspension and adherent culturesis described in WO97/37000, WO97/37001, WO03/023021, and WO03/023025. Inparticular, WO 03/023021 and WO 03/023025 describe laboratory andcommercial scale cell culture volumes of MDCK suspension cells inserum-free media, chemically defined media, and protein free media. Eachreference is incorporated herein in its entirety.

As an alternative to mammalian sources, cell lines for use in theinvention may be derived from avian sources such as chicken, duck,goose, quail or pheasant. Avian cell lines may be derived from a varietyof developmental stages including embryonic, chick and adult.Preferably, the cell lines are derived from the embryonic cells, such asembryonic fibroblasts, germ cells, or individual organs, includingneuronal, brain, retina, kidney, liver, heart, muscle, or extraembryonictissues and membranes protecting the embryo. Examples of avian celllines include avian embryonic stem cells (WO01/85938 and WO03/076601)and duck retina cells (WO2005/042728). Suitable avian embryonic stemcells, include the EBx cell line derived from chicken embryonic stemcells, EB45, EB14, and EB14-074 (WO2006/108846). Chicken embryofibroblasts (CEF) may also be used. These avian cells are particularlysuitable for growing influenza viruses.

Insect cell expression systems, such as baculovirus recombinantexpression systems, are known to those of skill in the art and describedin, e.g., Summers and Smith, Texas Agricultural Experiment StationBulletin No. 1555 (1987). Materials and methods for baculovirus/insertcell expression systems are commercially available in kit form from,inter alia, Invitrogen, San Diego Calif. Insect cells for use withbaculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Recombinant expression of proteins may also be conducted in bacterialhosts such as Escherichia coli, Bacillus subtilis, and Streptococcusspp. Yeast hosts suitable for recombinant expression of proteins includeSaccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenualpolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichiaguillerimondii, Pichia pastoris, Schizosaceharomyces pombe and Yarrowialipolytica.

Culture conditions for the above cell types are well-described in avariety of publications, or alternatively culture medium, supplements,and conditions may be purchased commercially, such as for example, asdescribed in the catalog and additional literature of CambrexBioproducts (East Rutherford, N.J.).

In certain embodiments, the host cells used in the methods describedherein are cultured in serum free and/or protein free media. A medium isreferred to as a serum-free medium in the context of the presentinvention in which there are no additives from serum of human or animalorigin. Protein-free is understood to mean cultures in whichmultiplication of the cells occurs with exclusion of proteins, growthfactors, other protein additives and non-serum proteins, but canoptionally include proteins such as trypsin or other proteases that maybe necessary for viral growth. The cells growing in such culturesnaturally contain proteins themselves.

Known serum-free media include Iscove's medium, Ultra-CHO medium (BioWhittaker) or EX-CELL (JRH Bioscience). Ordinary serum-containing mediainclude Eagle's Basal Medium (BME) or Minimum Essential Medium (MEM)(Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium(DMEM or EDM), which are ordinarily used with up to 10% fetal calf serumor similar additives. Optionally, Minimum Essential Medium (MEM) (Eagle,Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM orEDM) may be used without any serum containing supplement. Protein-freemedia like PF-CHO (JHR Bioscience), chemically-defined media like ProCHO4CDM (Bio Whittaker) or SMIF 7 (Gibco/BRL Life Technologies) andmitogenic peptides like Primactone or Pepticase (all from QuestInternational) or lactalbumin hydrolyzate (Gibco and othermanufacturers) are also adequately known in the prior art. The mediaadditives based on plant hydrolyzates have the special advantage thatcontamination with viruses, mycoplasma or unknown infectious agents canbe ruled out.

Cell culture conditions (temperature, cell density, pH value, etc.) arevariable over a very wide range owing to the suitability of the cellline employed according to the invention and can be adapted to therequirements of particular virus growth conditions or recombinantexpression details.

Cells may be grown in various ways e.g. in suspension, in adherentculture, on microcarriers.

Cells may be grown below 37° C. (e.g. 30-36° C.) during viralreplication (WO97/37001).

The method for propagating virus in cultured cells generally includesthe steps of inoculating the cultured cells with the strain to becultured, cultivating the infected cells for a desired time period forvirus propagation, such as for example as determined by virus titer orantigen expression (e.g. between 24 and 168 hours after inoculation) andcollecting the propagated virus. The cultured cells may be inoculatedwith a virus (measured by PFU or TCID₅₀) to cell ratio of 1:500 to 1:1,preferably 1:100 to 1:5, more preferably 1:50 to 1:10. The virus can beadded to a suspension of the cells or applied to a monolayer of thecells, and the virus is absorbed on the cells for at least 60 minutesbut usually less than 300 minutes, preferably between 90 and 240 minutesat 25° C. to 40° C., preferably 28° C. to 37° C.

The infected cell culture (e.g. monolayers) may be removed either byfreeze-thawing or by enzymatic action to increase the viral content ofthe harvested culture supernatants. The harvested fluids are then eitherinactivated or stored frozen. Cultured cells may be infected at amultiplicity of infection (“m.o.i.”) of about 0.0001 to 10, preferably0.002 to 5, more preferably to 0.001 to 2. Still more preferably, thecells are infected at a m.o.i of about 0.01. Infected cells may beharvested 30 to 60 hours post infection. Preferably, the cells areharvested 34 to 48 hours post infection. Still more preferably, thecells are harvested 38 to 40 hours post infection. Proteases (typicallytrypsin) are generally added during cell culture to allow viral release,and the proteases can be added at any suitable stage during the culture.

The vaccine compositions of the invention will generally be formulatedin a sub-virion form e.g. in the form of a split virus, where the virallipid envelope has been dissolved or disrupted, or in the form of one ormore purified viral proteins. The vaccine composition will contain asufficient amount of the antigen(s) to produce an immunological responsein the patient.

Methods of splitting viruses, such as influenza viruses, are well knownin the art e.g. see WO02/28422, WO02/067983, WO02/074336, WO01/21151,etc. Splitting of the virus is carried out by disrupting or fragmentingwhole virus, whether infectious (wild-type or attenuated) ornon-infectious (e.g. inactivated), with a disrupting concentration of asplitting agent. Splitting agents generally include agents capable ofbreaking up and dissolving lipid membranes, typically with a hydrophobictail attached to a hydrophilic head. The most preferred splitting agentis cetyltrimethylammoniumbromide (CTAB). Further details of splittingare given below in the context: of influenza viruses.

The disruption results in a full or partial solubilization of the virusproteins, altering the integrity of the virus. Preferred splittingagents are non-ionic and ionic (e.g. cationic) surfactants e.g.alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines,betains, polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg,alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds,sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butylphosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin,lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols(e.g. the Triton surfactants, such as Triton X-100 or Triton N101),polyoxyethylene sorbitan esters (the Tween surfactants), polyoxyethyleneethers, polyoxyethylene esters, etc.

Methods of purifying individual proteins from viruses are well known andinclude, for example, filtration, chromatography, centrifugation stepsand hollow fiber elution. In one embodiment, the proteins are purifiedby ion exchange resin.

As a further alternative, the vaccine may include a whole virus e.g. alive attenuated whole virus or, preferably, an inactivated whole virus.Methods of inactivating or killing viruses to destroy their ability toinfect mammalian cells are known in the art. Such methods include bothchemical and physical means. Chemical means for inactivating a virusinclude treatment with an effective amount of one or more of thefollowing agents: detergents, formaldehyde, formalin, BPL, or UV light.Additional chemical means for inactivation include treatment withmethylene blue, psoralen, carboxyfullerene (C60) or a combination of anythereof. Other methods of viral inactivation are known in the art, suchas for example binary ethylamine, acetyl ethyleneimine, or gammairradiation. Preferably, the virus is inactivated with BPL.

Pharmaceutical Compositions

Compositions of the invention are pharmaceutically acceptable. Theyusually include components in addition to the antigens e.g. theytypically include one or more pharmaceutical carrier(s) and/orexcipient(s). A thorough discussion of such components is available inRemington: The Science and Practice of Pharmacy (Gennaro, 2000; 20thedition, ISBN: 0683306472).

Compositions will generally be in aqueous form.

The composition may include one or more preservatives, such asthiomersal or 2-phenoxyethanol. It is preferred, however, that thevaccine should be substantially free from (i.e. less than 5 μg/ml)mercurial material e.g. thiomersal-free (Banzhoff (2000) ImmunologyLetters 71:91-96; WO02/097072). Vaccines containing no mercury are morepreferred. Preservative-free vaccines are particularly preferred.

It is preferred to include a physiological salt, such as a sodium salte.g. to control tonicity. Sodium chloride (NaCl) is preferred, which maybe present at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. 6.5 and 7.5. A method of theinvention may therefore include a step of adjusting the pH of the bulkvaccine prior to packaging.

The composition is preferably sterile. The composition is preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. The composition is preferablygluten free.

Compositions of the invention may include detergent e.g. apolyoxyethylene sorbitan ester surfactant (known as ‘Tweens’), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate. The detergent may be present only attrace amounts.

The composition may include material for a single immunization, or mayinclude material for multiple immunizations (i.e. a ‘multidose’ kit).The inclusion of a preservative is useful in multidose arrangements. Asan alternative (or in addition) to including a preservative in multidosecompositions, the compositions may be contained in a container having anaseptic adaptor for removal of material.

Vaccines are typically administered in a dosage volume of about 0.5 ml,although a half dose (i.e. about 0.25 ml) may be administered tochildren.

Compositions and kits are preferably stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight.

Methods of Treatment, and Administration of Vaccines

Compositions of the invention are suitable for administration to animal(and, in particular, human) patients, and the invention provides amethod of raising an immune response in a patient, comprising the stepof administering a composition of the invention to the patient.

The invention also provides a kit or composition of the invention foruse as a medicament.

The invention also provides the use of immunogenic proteins derived froma virus propagated on cell culture, wherein said vaccine issubstantially free of residual functional cell culture DNA, in themanufacture of a medicament for raising an immune response in a patient.

The immune response raized by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses, neutralizing capability andprotection after viral vaccination are well known in the art. Forinfluenza virus, for instance, human studies have shown that antibodytiters against HA are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [Potter & Oxford (1979)Br Med Bull 35: 69-75].

Compositions of the invention can be administered in various ways. Themost preferred immunization route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection, intranasal, oral, intradermal, transcutaneous, transdermal,etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. The patient may be less than 1 year old, 1-5 yearsold, 5-15 years old, 15-55 years old, or at least 55 years old. Patientsmay be elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65years), the young (e.g. ≤5 years old), hospitalized patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, immunodeficient patients, patients who have taken anantiviral compound (e.g. an oseltamivir or zanamivir compound forinfluenza; see below) in the 7 days prior to receiving the vaccine,people with egg allergies and people travelling abroad. The vaccines arenot suitable solely for these groups, however, and may be used moregenerally in a population.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naïve patients. Multiple doseswill typically be administered at least 1 week apart (e.g. about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as abacterial vaccine, such as a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H. influenzae type bvaccine, a meningococcal conjugate vaccine (such as a tetravalentA-C-W135-Y vaccine), a pneumococcal conjugate vaccine, etc.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound effective against the virus of the vaccine. Where the vaccineis an influenza vaccine, for instance, the compound(s) may be aneuraminidase inhibitor (e.g. oseltamivir and/or zanamivir). Theseantivirals include a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or a5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral effective againstinfluenza is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate.

Host Cell DNA

Measurement of residual host cell DNA is within the normal capabilitiesof the skilled person. The total amount of residual DNA in compositionsof the invention is preferably less than 20 ng/ml e.g. ≤10 ng/ml, ≤5ng/ml, ≤1 ng/ml, ≤100 pg/ml, ≤10 pg/ml, etc. As described above,substantially all of this DNA is preferably less than 500 base pairs inlength.

The assay used to measure DNA will typically be a validated assay(Guidance for Industry: Bioanalytical Method Validation. U.S. Departmentof Health and Human Services Food and Drug Administration Center forDrug Evaluation and Research (CDER) Center for Veterinary Medicine(CVM). May 2001; Lundblad (2001) Biotechnology and Applied Biochemistry34:195-197). The performance characteristics of a validated assay can bedescribed in mathematical and quantifiable terms, and its possiblesources of error will have been identified. The assay will generallyhave been tested for characteristics such as accuracy, precision,specificity. Once an assay has been calibrated (e.g. against knownstandard quantities of host cell DNA) and tested then quantitative DNAmeasurements can be routinely performed. Three principle techniques forDNA quantification can be used: hybridization methods, such as Southernblots or slot blots (Ji et al. (2002) Biotechniques. 32:1162-7);immunoassay methods, such as the system disclosed in Briggs (Briggs(1991) J Parenter Sci Technol. 45:7-12; and quantitative PCR (Lahijaniet al. (1998) Hum Gene Ther. 9:1173-80). These methods are all familiarto the skilled person, although the precise characteristics of eachmethod may depend on the host cell in question e.g. the choice of probesfor hybridization, the choice of primers and/or probes foramplification, etc. The system from Molecular Devices is a quantitativeassay for picogram levels of total DNA, and has been used for monitoringlevels of contaminating DNA in biopharmaceuticals (Briggs (1991) supra).A typical assay involves non-sequence-specific formation of a reactioncomplex between a biotinylated ssDNA binding protein, aurease-conjugated anti-ssDNA antibody, and DNA. All assay components areincluded in the complete Total DNA Assay Kit available from themanufacturer. Various commercial manufacturers offer quantitative PCRassays for detecting residual host cell DNA e.g. Althea Technologies,etc. A comparison of a chemiluminescent hybridization assay and thetotal DNA system for measuring host cell DNA contamination of a humanviral vaccine can be found in Lokteff et al. (2001) Biologicals.29:123-32.

These various analytical methods may also be used for measuring thelength of residual host cell DNA. As mentioned above, the average lengthof residual host cell DNA, after treatment with the alkylating agent, ispreferably less than 500 base pairs, or even less than 200 base pairs.

In relation to canine cells in particular, such as MDCK cells, analysisof the genome reveals 13 coding sequences <500 bp in length, 3 sequences<200 bp and 1 sequence <100 bp. Thus fragmentation of DNA to <200 bpremoves substantially all coding sequences, and it is highly unlikelythat any fragment would actually correspond to one of the 3 genes aroundthat length (namely: secretin at 81 bp; PYY at 108 bp; and osteocalcinat 135 bp).

Adjuvants

Compositions of the invention may include an adjuvant, which canfunction to enhance the immune responses (humoral and/or cellular)elicited in a patient who receives the composition. The use of adjuvantswith viral vaccines is well known e.g. in hepatitis vaccines, poliovaccines, etc.

Adjuvants that can be used with the invention include, but are notlimited to, aluminum salts, immunostimulatory oligonucleotides,saponins, lipid A analogs (such as 3dMPL) and oil-in-water emulsions.These and other adjuvants are disclosed in more detail in Powell &Newman (Vaccine Design: The Subunit and Adjuvant Approach, Plenum Press1995, ISBN 0-306-44867-X) and O'Hagan (Vaccine Adjuvants: PreparationMethods and Research Protocols, volume 42 of Methods in MolecularMedicine series, ISBN: 1-59259-083-7).

The adjuvants known as aluminum hydroxide and aluminum phosphate may beused. These names are conventional, but are used for convenience only,as neither is a precise description of the actual chemical compoundwhich is present (e.g. see chapter 9 of Powell & Newman). The inventioncan use any of the “hydroxide” or “phosphate” adjuvants that are ingeneral use as adjuvants. Adsorption to these salts is preferred.

Immunostimulatory oligonucleotides with adjuvant activity are wellknown. They may contain a CpG motif (a dinucleotide sequence containingan unmethylated cytosine linked by a phosphate bond to a guanosine), aTpG motif, an oligo-dT sequence, an oligo-dC sequence, double-strandedRNA, palindromic sequences, a poly(dG) sequence, etc. Immunostimulatoryoligonucleotides will typically comprise at least 20 nucleotides, andmay comprise fewer than 100 nucleotides.

Saponins (chapter 22 of Powell & Newman) are a heterologous group ofsterol glycosides and triterpenoid glycosides that are found in thebark, leaves, stems, roots and even flowers of a wide range of plantspecies. Saponins from the bark of the Quillaia saponaria Molina treehave been widely studied as adjuvants. Saponin adjuvant formulationsinclude purified formulations, such as QS21, as well as lipidformulations, such as ISCOMs. Saponin compositions have been purifiedusing HPLC and RP-HPLC, and specific purified fractions using thesetechniques have been identified, including QS7, QS17, QS18, QS21, QH-A,QH-B and QH-C. Preferably, the saponin is QS21. A method of productionof QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulationsmay also comprise a sterol, such as cholesterol (WO96/33739).Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs), ISCOMs, whichtypically also include a phospholipid.

3dMPL (also known as 3 de-O-acylated monophosphoryl lipid A or3-O-desacyl-4′-monophosphoryl lipid A) is an adjuvant in which position3 of the reducing end glucosamine in monophosphoryl lipid A has beende-acylated. 3dMPL has been prepared from a heptoseless mutant ofSalmonella minnesota, and is chemically similar to lipid A but lacks anacid-labile phosphoryl group and a base-labile acyl group. 3dMPL cantake the form of a mixture of related molecules, varying by theiracylation (e.g. having 3, 4, 5 or 6 acyl chains, which may be ofdifferent lengths).

Various oil-in-water emulsions with adjuvant activity are known. Theytypically include at least one oil and at least one surfactant, with theoil(s) and surfactant(s) being biodegradable (metabolisable) andbiocompatible. The oil droplets in the emulsion generally havesub-micron diameters, with these small sizes being achieved with amicrofluidiser to provide stable emulsions. Droplets with a size lessthan 220 nm are preferred as they can be subjected to filtersterilization.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’, as        described in more detail in Chapter 10 of Powell & Newman and        chapter 12 of O'Hagan. The MF59 emulsion advantageously includes        citrate ions e.g. 10 mM sodium citrate buffer.    -   An emulsion of squalene, a tocopherol, and Tween 80. The        emulsion may include phosphate buffered saline. It may also        include Span 85 (e.g. at 1%) and/or lecithin. These emulsions        may have from 2 to 10% squalene, from 2 to 10% tocopherol and        from 0.3 to 3% Tween 80, and the weight ratio of        squalene:tocopherol is preferably ≤1 as this provides a more        stable emulsion. Squalene and Tween 80 may be present volume        ratio of about 5:2. One such emulsion can be made by dissolving        Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this        solution with a mixture of (5 g of DL-α-tocopherol and 5 ml        squalene), then microfluidising the mixture. The resulting        emulsion may have submicron oil droplets e.g. with an average        diameter of between 100 and 250 nm, preferably about 180 nm.    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL. The        emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 m/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL. The aqueous phase may contain a phosphate buffer.

Influenza Vaccines

The invention is particularly suitable for preparing influenza virusvaccines. Various forms of influenza virus vaccine are currentlyavailable e.g. see chapters 17 & 18 of Plotkin & Orenstein (Vaccines,4th edition, 2004, ISBN: 0-7216-9688-0). They are generally based eitheron live virus or on inactivated virus. Inactivated vaccines may be basedon whole virions, ‘split’ virions, or on purified surface antigens(including hemagglutinin). Influenza antigens can also be presented inthe form of virosomes (nucleic acid free viral-like liposomalparticles). Antigens purified from a recombinant host (e.g. in an insectcell line using a baculovirus vector) may also be used.

Chemical means for inactivating a virus include treatment with aneffective amount of one or more of the following agents: detergents,formaldehyde, β-propiolactone, methylene blue, psoralen,carboxyfullerene (C60), binary ethylamine, acetyl ethyleneimine, orcombinations thereof. Non-chemical methods of viral inactivation areknown in the art, such as for example UV light or gamma irradiation.

Virions can be harvested from virus-containing fluids by variousmethods. For example, a purification process may involve zonalcentrifugation using a linear sucrose gradient solution that includesdetergent to disrupt the virions. Antigens may then be purified, afteroptional dilution, by diafiltration.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N101, cetyltrimethylammonium bromide, etc.) toproduce subvirion preparations, including the ‘Tween-ether’ splittingprocess. Methods of splitting influenza viruses are well known in theart e.g. WO02/28422, WO02/067983, WO02/074336, WO01/21151, WO02/097072,WO2005/113756 etc. Splitting of the virus is typically carried out bydisrupting or fragmenting whole virus, whether infectious ornon-infectious with a disrupting concentration of a splitting agent. Thedisruption results in a full or partial solubilization of the virusproteins, altering the integrity of the virus. Preferred splittingagents are non-ionic and ionic (e.g. cationic) surfactants e.g.alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines,betains, polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg,alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds,sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butylphosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin,lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols(e.g. the Triton surfactants, such as Triton X-100 or Triton N101),polyoxyethylene sorbitan esters (the Tween surfactants), polyoxyethyleneethers, polyoxyethlene esters, etc. One useful splitting procedure usesthe consecutive effects of sodium deoxycholate and formaldehyde, andsplitting can take place during initial virion purification (e.g. in asucrose density gradient solution). Thus a splitting process can involveclarification of the virion-containing material (to remove non-virionmaterial), concentration of the harvested virions (e.g. using anadsorption method, such as CaHPO₄ adsorption), separation of wholevirions from non-virion material, splitting of virions using a splittingagent in a density gradient centrifugation step (e.g. using a sucrosegradient that contains a splitting agent such as sodium deoxycholate),and then filtration (e.g. ultrafiltration) to remove undesiredmaterials. Split virions can usefully be resuspended in sodiumphosphate-buffered isotonic sodium chloride solution.

Purified surface antigen vaccines comprise the influenza surfaceantigens hemagglutinin and, typically, also neuraminidase. Processes forpreparing these proteins in purified form are well known in the art.

Influenza virus strains for use in vaccines change from season toseason. In the current inter-pandemic period, vaccines typically includetwo influenza A strains (H1N1 and H3N2) and one influenza B strain, andtrivalent vaccines are typical. The invention may also use HA frompandemic strains (i.e. strains to which the vaccine recipient and thegeneral human population are immunologically naïve), such as H2, H5, H7or H9 subtype strains (in particular of influenza A virus), andinfluenza vaccines for pandemic strains may be monovalent or may bebased on a normal trivalent vaccine supplemented by a pandemic strain.Depending on the season and on the nature of the antigen included in thevaccine, however, the invention may protect against one or more ofinfluenza A virus hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15 or H16.

As well as being suitable for immunizing against inter-pandemic strains,the compositions of the invention are particularly useful for immunizingagainst pandemic strains. The characteristics of an influenza strainthat give it the potential to cause a pandemic outbreak are: (a) itcontains a new hemagglutinin compared to the hemagglutinins incurrently-circulating human strains, i.e. one that has not been evidentin the human population for over a decade (e.g. H2), or has notpreviously been seen at all in the human population (e.g. H5, H6 or H9,that have generally been found only in bird populations), such that thehuman population will be immunologically naïve to the strain'shemagglutinin; (b) it is capable of being transmitted horizontally inthe human population; and (c) it is pathogenic to humans. A virus withH5 hemagglutinin type is preferred for immunizing against pandemicinfluenza, such as a H5N1 strain. Other possible strains include H5N3,H9N2, H2N2, H7N1 and H7N7, and any other emerging potentially pandemicstrains. Within the H5 subtype, a virus may fall into HA clade 1, HAclade 1′, HA clade 2 or HA clade 3 (World Health Organization (2005)Emerging Infectious Diseases 11(10):1515-21), with clades 1 and 3 beingparticularly relevant. Other strains whose antigens can usefully beincluded in the compositions are strains which are resistant toantiviral therapy (e.g. resistant to oseltamivir and/or zanamivir),including resistant pandemic strains.

Compositions of the invention may include antigen(s) from one or more(e.g. 1, 2, 3, 4 or more) influenza virus strains, including influenza Avirus and/or influenza B virus. A trivalent vaccine is preferred,including antigens from two influenza A virus strains and one influenzaB virus strain.

The influenza virus may be a reassortant strain, and may have beenobtained by reverse genetics techniques. Thus an influenza A virus mayinclude one or more RNA segments from a A/PR/8/34 virus (typically 6segments from A/PR/8/34, with the HA and N segments being from a vaccinestrain, i.e. a 6:2 reassortant). It may also include one or more RNAsegments from a A/WSN/33 virus, or from any other virus strain usefulfor generating reassortant viruses for vaccine preparation. Typically,the invention protects against a strain that is capable ofhuman-to-human transmission, and so the strain's genome will usuallyinclude at least one RNA segment that originated in a mammalian (e.g. ina human) influenza virus. It may include NS segment that originated inan avian influenza virus.

HA is the main immunogen in current inactivated influenza vaccines, andvaccine doses are standardized by reference to HA levels, typicallymeasured by SRID. Existing vaccines typically contain about 15 μg of HAper strain, although lower doses can be used e.g. for children, or inpandemic situations, or when using an adjuvant. Fractional doses such as½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ have been used, as have higherdoses (e.g. 3× or 9× doses). Thus vaccines may include between 0.1 and150 μg of HA per influenza strain, preferably between 0.1 and 50 μg e.g.0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc. Particulardoses include e.g. about 15, about 10, about 7.5, about 5, about 3.8,about 1.9, about 1.5, etc. per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10⁶⁵-10^(7.5)) per strain is typical.

HA used with the invention may be a natural HA as found in a virus, ormay have been modified. For instance, it is known to modify HA to removedeterminants (e.g. hyper-basic regions around the cleavage site betweenHA1 and HA2) that cause a virus to be highly pathogenic in avianspecies.

After treating a virion-containing composition to degrade host cell DNA(e.g. with BPL), the degradation products are preferably removed fromvirions e.g. by anion exchange chromatography.

Once an influenza virus has been purified for a particular strain, itmay be combined with viruses from other strains e.g. to make a trivalentvaccine as described above. It is preferred to treat each strainseparately and to mix monovalent bulks to give a final multivalentmixture, rather than to mix viruses and degrade DNA from a multivalentmixture.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x±10%.

By the terms “isolated” and “purified” is meant at least 50% pure, morepreferably 60% pure (as with most split vaccines), more preferably 70%pure, or 80% pure, or 90% pure, or 95% pure, or greater than 99% pure.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Where a cell substrate is used for reassortment or reverse geneticsprocedures, it is preferably one that has been approved for use in humanvaccine production e.g. as in Ph Eur general chapter 5.2.3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the results of PCR amplification of 6 named targetsin the MDCK genome. Some samples were diluted as indicated, up to1:10000, prior to PCR.

FIG. 3 shows the results of PCR amplification of SINE sequences in theMDCK genome. As in FIGS. 1 & 2, DNA was sometimes diluted prior to PCR.In the lower panel, the figures are the starting amount of DNA in thePCR (fg).

FIG. 4 shows the effect of BPL treatment on the size of MDCK DNA.Genomic DNA was incubated with BPL at different temperatures and fordifferent lengths of time. FIG. 5 shows similar results. Agarose gelswere stained with SYBRGold.

FIG. 6 shows capillary electrophoresis of residual DNA (top line)relative to markers (bottom line) having the indicated sizes.

MODES FOR CARRYING OUT THE INVENTION

Influenza viruses (A/New Caledonia/20/99(H1N1), A/Panama/2007/99(H3N2);B/Jiangsu/10/2003; A/Wyoming/3/2003(H3N2)) were grown in MDCK cells in asuspension culture, following the teaching of WO97/37000, WO03/23025 andWO04/92360. The final culture medium was clarified to provide virions,which were then subjected to chromatography andultrafiltration/diafiltration. Virions in the resulting material wereinactivated using β-propiolactone (final concentration 0.05% v/v;incubated for 16-20 hours at 2-8° C., and then hydrolyzed by incubatingat 37° C. for 2-2.5 hours). CTAS was then used to split the virions, andvarious further processing steps gave a final monovalent bulk vaccinecontaining purified surface proteins.

MDCK DNA was characterized to evaluate its amount, size, and integrityat three stages of the manufacturing process: (A) after theultrafiltration/diafiltration step; (B) after β-propiolactone treatment;and (C) in the final monovalent bulk. Capillary gel electrophoresis andnucleic acid amplification were used to investigate the size, theintegrity and the biological activity of any residual genomic DNA.

Size Determination

As mentioned above, the size of residual host cell DNA was analyzed atstages (A), (B) and (C) by capillary gel electrophoresis. The analysiswas performed on five separate virus cultures.

500 μl samples were removed at these three points and treated with 10 μlproteinase K at 56° C. for 16 to 22 h followed by a total DNA extractionwith the DNA Extractor Kit (Wako Chemicals) following the manufacturer'sinstructions. DNA was resuspended in 500 μl ultra pure water forelectrophoresis on a P/ACE MDQ Molecular Characterization System(Beckman Coulter) at a constant temperature of 20° C. Eleven molecularsize markers were used, from 72 to 1353 bp. The nucleic acid detectionlimit (DL) for this method was 0.7 μg/ml.

Based on the size markers and relative density of the DNA bands, thedistribution and size of the DNA fragments were determined and assignedto four different size categories between ranging from <200 bp to >1000bp. FIG. 6 shows capillary electrophoresis of a stage (C) sample. Theanalysis shows that all detectable residual DNA in this sample issubstantially below 200 bp in length.

Average results from the five cultures are shown in the following table:

Stage DNA amount <200 bp 200-500 bp 500-1000 bp >1000 bp A 47 mg 25% 12%6% 55% B 5 mg 79% 18% 3%  4% C 0.07 mg >99%  <DL <DL <DL

BPL treatment thus causes a ˜10-fold reduction in the amount of DNA, butalso shifts the distribution away from long sequences towards smallfragments <200 bp. Further processing, between steps (B) and (C),including chromatography and ultrafiltration steps, reduced total DNAlevels another ˜70-fold, and removed all detectable DNA ≥200 bp.

DNA Amplification

Neoplastic cell transformation is a phenomenon often associated withmodified proto-oncogenes and/or modified tumor suppressor genes.Sequences from several such canine genes were analyzed by PCR before andafter BPL treatment i.e. at points (A) and (B). In addition, DNA fromuninfected MDCK cells was treated and tested. Proto-oncogenes testedwere: H-ras and c-myc. Tumor suppressor genes tested were: p53;p21/waf-1; and PTEN. In addition, repetitive SINE sequences wereanalyzed by PCR. The high copy number for SINES facilitates sensitivedetection.

All samples were spiked with an external control DNA (pUC19 fragment)for monitoring the quality of the sample preparation and the PCR. In allexperiments a consistent amplification of the spike control wasobserved, indicating that residual hydrolyzed BPL and by-productspresent in the PCR mixtures does not have inhibitory effects on theassay and ensured a sufficient quality of sample preparation and PCR.Samples were diluted in 10-fold steps before amplification, until PCRproducts could not be detected, thereby indicating the log reduction inDNA levels. The detection limit for the PCR assay is 55 pg.

PCR products were analyzed by agarose gel electrophoresis. FIG. 1 showsresults obtained in uninfected MDCK cells, and FIG. 2 shows resultsobtained during virus culture. All six analyzed genes showed a strongamplification signal before BPL treatment, but signal strengthdiminished after exposure to BPL. In all tested production lots,residual MDCK genomic DNA was decreased by at least 2 log values afterBPL exposure.

Because FIG. 2 shows results at stage (B), before further purification,the results over-represent DNA present in final bulk vaccines. Due tothe sensitivity when using SINE sequences, however, PCR was alsopossible at stage (C), in the final monovalent bulk. Results of thisanalysis are shown in FIG. 3. DNA amplification in (A) and (B) wasrelatively similar for the SINE region, indicating that BPL is lessactive on smaller DNA sequences. Even for these small sequences,however, there was a notable reduction in the amplification signal forall of the analyzed samples. Comparison of the PCR signal intensitiesbetween sample collection point (B) and (C) reflects the reduction ofresidual DNA due to intermediate purification steps.

Extrapolating from the gene-based analysis, and based directly on theSINE-based analysis, a total DNA level of <1 ng per dose, and often <100pg per dose, is expected in a final vaccine.

Temperature

BPL treatment during virus inactivation had two independent steps: (1)add BPL to the virion-containing mixture at 2-8° C.; then (2) raise thetemperature to 37° C. to hydrolyse BPL. The effects of the two BPL stepson MDCK DNA inactivation and fragmentation were investigated.

Purified genomic DNA from uninfected MDCK cells was treated with a finalconcentration of 0.05% (v/v) BPL at 2-8° C. for 16 hours, or at 37° C.or 50° C. for up to 6.5 hours. Fragmentation of DNA was checkedafterwards, and results are shown in FIG. 4. These experiments revealthe activity of BPL against cellular DNA, but uninfected cells do notreflect the state of MDCK after viral growth because cellular DNA isalready highly fragmented due to apoptosis.

At 2-8° C., the difference between untreated and treated DNA on anagarose gel was negligible, suggesting that DNA fragmentation by BPL maynot substantially occur during under these conditions. In contrast, DNAwas greatly modified at both 37° C. and 50° C., with the highertemperature leading to an accelerated reaction kinetic. DNA fromuntreated cells showed a relatively distinct band in an agarose gel,with a light smear of degradation products (FIG. 5, lane 3). After ashort BPL incubation at 37° C., however, MDCK DNA smeared down from thegel slots and band signal intensity decreased (FIG. 5, lane 2). Longerincubation periods resulted in the disappearance of large genomic DNAmolecules and enhanced fragmentation of MDCK DNA.

In further experiments, BPL was first incubated with cells at 4° C. for16 hours. In a first population, the temperature was raized to 37° C.for 2 hours; in a second population, BPL was removed by centrifugalfilters and then the temperature was raized. Much lower (>2 log lower)residual DNA levels were seen in the first population.

Thus the DNA fragmentation observed during BPL treatment seems to occurmainly during the BPL hydrolysis step at 37° C. rather than during thevirus inactivation step at 2-8° C.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

The invention claimed is:
 1. A vaccine comprising immunogenic proteinsderived from two or more influenza virus strains propagated on cellculture, wherein the vaccine comprises 10 pg/mL to less than 20 ng/mL ofresidual cell culture DNA, wherein the length of the residual cellculture DNA is less than 200 base pairs, and wherein the cell culture isa mammalian cell culture or an avian cell culture.
 2. The vaccine ofclaim 1, comprising less than 0.1% β-propiolactone (BPL).
 3. The vaccineof claim 1, wherein the cell culture is selected from the groupconsisting of Madin Darby canine kidney (MDCK) cells, Vero cells, andhuman embryonic retinoblasts.
 4. The vaccine of claim 1, furthercomprising an oil-in-water emulsion adjuvant.
 5. The vaccine of claim 4,wherein the oil-in-water emulsion adjuvant comprises oil droplets havinga sub-micron diameter.
 6. The vaccine of claim 5, wherein theoil-in-water emulsion adjuvant comprises squalene.
 7. The vaccine ofclaim 1, wherein the immunogenic proteins are viral antigens selectedfrom the group consisting of hemagglutinin (HA), neuraminidase (NA),nucleoprotein (NP), matrix protein (M1), membrane protein (M2), and oneor more of the transcriptase components (PB1, PB2 and PA).
 8. Thevaccine of claim 7, wherein the immunogenic proteins are NA.
 9. Thevaccine of claim 7, wherein the immunogenic proteins are HA.
 10. Thevaccine of claim 9, comprising about 7.5 μg, about 10 μg, or about 15 μgHA per influenza virus strain in a single dosage volume of about 0.5 mL.11. The vaccine of claim 1, comprising immunogenic proteins derived fromthree or more influenza virus strains propagated on cell culture. 12.The vaccine of claim 1, wherein the length of the residual cell cultureDNA is from 20 to 200 base pairs.
 13. The vaccine of claim 1, whereinthe length of the residual cell culture DNA is about 72 base pairs.